hTERT-HME1

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SOP: Thawing, Propagating and Cryopreserving NCI-PBCF-CRL4010 (hTERT-HME1) cells Subculture Medium Subculture Method Viable Cells/mL/Cryovial Cryopreservation Medium 0.25 % (w/v) trypsin-0.53 mM EDTA (ATCC cat no. 30-2101). Subculturing reagents should be pre-warmed before use by placing into a water bath set at is 35 o C to 37 o C for 15 min to 30 min. After 30 min, the subculturing medium should be moved to room temperature until used. Subculturing reagents should be stored at 2 o C to 8 o C when not in use. The attached hTERT-HME1 cells are subcultured using 0.25 % (w/v) trypsin-0.53 mM EDTA (ATCC cat no. 30-2101). The enzymatic action of the trypsin-EDTA is stopped by adding 0.1% (w/v) Soybean Trypsin Inhibitor to the detached cells. A split ratio of 1:8 to 1:10 or a seeding density of 1 x 10 4 viable cells/cm 2 to 8 x 10 3 viable cells/cm 2 is used when subculturing hTERT-HME1 cells. The target number of viable cells/mL/cryovial is: 2.0 x 10 6 (acceptable range: 2 x 10 6 viable cells/mL to 3 x 10 6 viable cells/mL). The cryopreservation medium for hTERT-HME1 cells is complete growth medium (MEGM) containing 10 % (v/v) DMSO (ATCC cat no. 4-X). General Procedure to be applied throughout the SOP Aseptic Technique Traceability of material/reagents Expansion of cell line Use of good aseptic technique is critical. Any materials that are contaminated, as well as any materials with which they may have come into contact, must be disposed of immediately. Record the manufacturer, catalog number, lot number, date received, date expired and any other pertinent information for all materials and reagents used. Record information in the Reagent Lot Traceability Table 4 (Appendix 6). Record the subculture and growth expansion activities, such as passage number, % confluence, % viability, cell morphology (see Figures 1,2,3 Appendix 1) and population doubling levels (PDLs), in the table for Cell Expansion (Table 5, Appendix 6). Calculate PDLs using the equation in Appendix 7. Medium volumes Medium volumes are based on the flask size as outlined in Table 1. Glossary of Terms Refer to Glossary of Terms used throughout the document (see Appendix 4). Safety Precaution Refer to Safety Precautions pertaining to the propagation and cryopreservation of hTERT-HME1 (See Appendix 8). Page 4 of 24
SOP: Thawing, Propagating and Cryopreserving NCI-PBCF-CRL4010 (hTERT-HME1) cells Table 1: Medium Volumes Flask Size Medium Volume Range 12.5 cm 2 (T-12.5) 3 mL to 6 mL 25 cm 2 (T-25) 5 mL to 13 mL 75 cm 2 (T-75) 10 mL to 38 mL 150 cm 2 (T-150) 30 mL to 75 mL 175 cm 2 (T-175) 35 mL to 88 mL 225 cm 2 (T-225) 45 mL to 113 mL 3. Reagents Follow Product Information Sheet storage and/or thawing instructions. Below is a list of reagents for the propagation, subcultivation and cryopreservation of hTERT-HME1 cells. Table 2: Reagents for Expansion, Subculturing and Cryopreservation of hTERT-HME1 cells Complete growth medium reagents MEGM (Mammary Epithelial Growth Medium) Lonza/Clonetics cat no. CC-3150) Subculturing reagents Trypsin-EDTA (0.25 % (w/v) Trypsin/0.53 mM EDTA ) (ATCC cat no. 30-2101) Dulbecco’s Phosphate Buffered Saline (DPBS); modified without calcium chloride and without magnesium chloride (ATCC cat no.30-2200) Cryopreservation medium reagents MEGM (Mammary Epithelial Growth Medium) Lonza/Clonetics cat no. CC-3150) 10% (v/v) Dimethyl Sulfoxide (DMSO) (ATCC cat no.4-X) 0.1% (w/v) Soybean Trypsin Inhibitor (ATCC cat no.30-2104) Page 5 of 24
Page 1 and 2: NCI-PBCF-CRL4010 (hTERT-HME1) Mamma
Page 3: SOP: Thawing, Propagating and Cryop
Page 7 and 8: SOP: Thawing, Propagating and Cryop
Page 23 and 24: Table 5: Cell Expansion FROM FLUID

hTERT-HME1

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